The tremendous reduction in global morbidity and mortality achieved through world-wide immunization programs relies directly upon the capacity to manufacture sufficient vaccines at an affordable price, especially in the developing world, and upon maintaining the vaccine potency up to the time of delivery, even under extreme environmental conditions (Duclos et al. 2009).
Most vaccines are currently stored and distributed in freeze-dried (lyophilized) form. At the point of delivery, lyophilized vaccines must be reconstituted with diluent, typically sterile water, that is shipped with the vaccine. Most of these vaccines are delivered by injection with a syringe and needle. The major drawbacks of this method include needle-stick injuries to health care workers (Panlilio et al. 2004), needle-phobia and discomfort for patients facing increasingly crowded immunization schedules (Miller and Pisani 1999), and the costs and complexity of safe disposal of sharps in the medical waste stream. In the developing world, unsafe injection practices such as reuse of needles represent a risk to patients many times higher than needle-stick injuries to health care workers (Gyawali et al. 2013). An estimated 16 billion injections are given annually in the developing world. Unsafe injections were estimated in 1999 to cause 1.3 million deaths annually and to cost more than $535 million to treat bloodborne diseases transmitted by unsafe injection. It is estimated that unsafe injections infect more than 88,000 people with HIV annually (Hutin et al. 2003).
There are many possible solutions to resolving the global problem of needle-stick injuries and unsafe injections, including reducing unnecessary injections, improving injection practices, improving sharps waste management and developing and using safer injection devices. A more radical solution is to move away from syringes and needles as delivery systems all together and toward delivery of essential agents like vaccines via needle-free methods, such as transdermal or mucosal delivery.
Mucosa is considered to be one of the largest barriers to infection in the human body. For this reason, mucosal administration of antigens (or vaccines) can theoretically be used to induce mucosal response toward systemic protection from infection at a variety of mucosal sites in the body. Mucosal routes such as oral intestinal, oral buccal, oral sublingual, nasal, ocular, pulmonary, rectal, and vaginal administration provide excellent opportunities for the delivery of a variety of dry preserved vaccines without a need for pre-delivery reconstitution. Skin vaccination also offers immunologic advantages due to vaccine targeting to antigen-presenting cells of the skin, as well as access to draining lymph nodes (Glenn et al. 2006). Micro-needle patches placed on the skin enable reliable vaccine targeting to the skin using a device that is easy to administer and is compatible with dry carbohydrate-glass formulations that require storage in a dry state (Prausnitz et al. 2009).
Currently many conventional pharmaceuticals can be stored at ambient temperatures (AT) and delivered via oral (intestinal, sublingual, and buccal), transdermal, respiratory, vaginal, and anal delivery routes without reconstitution with water before delivery, avoiding painful parenteral injection and necessity of medical personnel. To achieve needle-free delivery, the pharmaceutical industry has developed sophisticated methods and tools for production: tablets, dissolvable films, patches, suppositories, ointment, creams, and capsules (including enteric coated capsules for intestinal delivery). These methods have been broadly described in the literature (Guidice 2006, O'Hagan 2004), however none to date have been effectively applied to vaccines and other fragile biopharmaceuticals (therapeutic proteins, probiotics, etc.).
A primary reason these methods have not been used with vaccines is because conventional preservation techniques (i.e. freeze-drying and spray-drying) have failed to deliver potent ambient-temperature stable products that can survive both the harmful conditions needed for preparing devices used for needle-free delivery, and storage (including distribution) at ambient temperatures.
Drying Technologies
Drying is required for formulation of ambient-temperature stable vaccines.
Stabilization of vaccines to enable storage at room or higher temperatures (i.e. 37° C.) can be achieved in a partially dehydrated state only for a limited amount of time (several days); however, long-term stabilization of vaccines requires arresting molecular mobility to stop the degradation processes that occur during storage. It is currently recognized that one of the only ways this can be achieved is by immobilization of biologicals in carbohydrate glasses, or vitrification: the transformation from a liquid into a supercooled or supersaturated, noncrystalline, amorphous solid state, known as the “glass state.” In general terms glasses are thermodynamically unstable, amorphous materials, however, they can be very stable for long periods of time because of their very high viscosity. For example, a typical liquid has a flow rate of 10 m/s compared to 10-14 m/s in the glass state.
The basic premise of this work is that the high viscosity of the glass state will arrest all diffusion-limited physical processes and chemical reactions, including the processes responsible for the degradation of biological materials. This premise is based on Einstein's theory that establishes the inverse proportionality between viscosity and molecular mobility (or diffusion coefficients of molecules). The presence of water in a sample has a strong plasticizing effect, which decreases the glass transition temperature (Tg) and thus limits stability at higher temperatures. For example, for an 80% sucrose solution, Tg is about −40° C. while the Tg of a 99% solution is about 52° C. Therefore, if specimens are to be preserved without degradation in the glass state at an ambient temperature, they must be strongly dehydrated.
Dehydration (drying) can be very damaging to vaccines and other biologicals if performed in the absence of protective, glass-forming carbohydrates (i.e. sucrose, mannitol, etc.). These molecules replace water of hydration at the surface of biological molecules, and this way protects the biologicals from destruction associated with hydration forces that arise during dehydration.
Freeze Drying
Freeze-drying (FD) has failed to deliver ambient-temperature stable vaccines.
Despite its limitations and shortcomings, freeze-drying has remained, for more than 50 years, the primary method to stabilize fragile biopharmaceuticals in the dry state. This is in part because conventional belief suggests that drying at low temperatures would be less damaging, and, in part because for many years there were no alternative drying technologies available that were scalable and maintained product integrity. Currently available lyophilized vaccines must be shipped and stored in a “cold chain” to maintain vaccine potency, deviation from which can result in incapacitating losses in vaccine titer.
Freeze-drying can also be very damaging, with lyophilization-induced injury happening both during freezing and during subsequent ice sublimation from frozen specimens at intermediate low temperatures (between −50° C. and −20° C.) at which most damaging cryochemical reactions occur.
To produce micronized powders for pulmonary or respiratory delivery, FD vaccines require milling. Although it has been demonstrated that FD measles vaccine can be micronized using a jet mill with only a small loss of activity titer (LiCalsi et al. 2001), the reported efficacy for milled freeze-dried measles vaccine is much below that for nebulized liquid-reconstituted measles vaccine (de Swart et al. 2007). As de Swart suggested (de Swart et al. 2006), this could be due to the inherently low stability of freeze-dried vaccines, which are then damaged further in the milling process.
Spray Drying
Spray-drying (SD) has failed to deliver ambient temperature stable vaccines.
Other scientific groups have avoided use of freeze-drying and turned to spray-drying (SD) in order to obtain dry microspheres suitable for respiratory delivery without involvement of a milling process, which requires special equipment and containment. Unfortunately, conventional spray-drying involves spraying (formation of small drops) of a liquid to be dried (usually water-based) into hot (typically 90° C. or above) air, which quickly evaporates water from the drops and sterilizes the material in the process. To avoid the damaging effects of high temperature, the spray-drying process should be modified to decrease the maximum temperature of the drops of vaccine during the process. This can be achieved by decreasing the temperature of the air and the drops simultaneously, and reducing the diameter of the drops of vaccine that that are produced by the spraying nozzle.
Aktiv-Dry LLC has been the leading group to use a spray-drying approach for preparation of measles vaccine for pulmonary delivery. Aktiv Dry has used supercritical CO2 to decrease the diameter of drops sprayed into air simultaneously with decrease of the air temperature. They have reported that the lower temperature SD process allows them to produce micronized vaccines with good activity after drying. However, their vaccines are not stable at ambient temperatures and lose more than 0.5 logs (>70%) of activity after only one week storage at 37° C., which is worse than the stability of measles vaccine currently produced by freeze-drying.
There are fundamental reasons (some of them are addressed below) explaining why it is very difficult and may be even impossible to achieve both good initial yield and stability of live vaccines that have been dried via SD. For one, it is very difficult to remove water from spray-dried particles which contain sugars or other glass forming molecules if during spray drying the temperature inside drops did not increase above the vaccine damaging level. This is because the rate of evaporative drying is limited by water mobility inside the drop and it becomes very slow when the drop loses most of its water and becomes very viscous. It is well known that the characteristic time (t) of the diffusion relaxation in the drop with diameter (d) is about t=d2/D, where D is the water diffusion coefficient and d is the drop diameter. In dilute solutions, D=105 sm2/sec and t=0.1 sec for small drops with diameter d=10μ. However, in drops containing concentrated solutions (syrups), it will greatly increase with a decrease of molecular mobility and diffusion coefficient (D). In concentrated syrups, D is larger than 105 sm2/sec by many orders of magnitude, which makes t many orders of magnitude higher than the typical spray-drying process time. Thus, a significant amount of water, resulting in a high mobility, will remain in the drops after spray-drying if the drops contain sugars or other glass forming additives required to protect the vaccine from the desiccation stress until spray drying is performed at temperatures that are substantially higher than the glass transition temperatures of protective carbohydrates in the drops. Spay drying at high temperatures normally inactivate vaccines and other biopharmaceuticals. Conventionally, spray drying was used as a disinfective process for milk and many other products. Thus, during spray-drying, decreasing the air temperature is necessary to avoid vaccine inactivation; yet, that will result in a higher concentration of water remaining in the material after the drying process, which will negatively affect stability during subsequent storage at ambient temperatures.
Both spray-drying and freeze-drying had been used for more than 50 years and attempts to apply these technologies to produce ambient temperature stable live attenuated vaccines and other fragile biopharmaceuticals had been unsuccessful.
A scalable Foam Drying under vacuum technology as described in U.S. Pat. No. 5,766,520 was introduced by Dr. Bronshtein as an alternative to freeze-drying and spray drying to produce thermostable biopharmaceuticals. In other words, foam drying was introduced to scale up the film drying. This foam drying technology called “Preservation by Foam Formation (PFF)” has many drawback including uncontrollable eruptions, difficulties of the process control and reproducible execution.
Preservation by Vaporization (PBV)—State of the Art of Foam Drying
In 2004, Dr. Bronshtein proposed Preservation by Vaporization (PBV) technology, during which a partially frozen material (slush) sublimates, boils and evaporates simultaneously (PCT Patent Application WO/2005117962). PBV is scalable, easy to control and reproduce, and has minimum splashing. Preliminary studies have illustrated the unique benefits of PBV technology, including:
higher activity titer after drying and thermostability during subsequent storage at ambient temperature (increased shelf-life);
eliminates the need for using a “cold chain”;
allows subsequent mechanical and jet milling (micronization) with minimum activity loss;
allows drying of vaccines encapsulated in gel microparticles for better intestinal delivery, avoiding the need for pre-delivery reconstitution with water; and
allows short-term (several hours) stability at 60° C. to 90° C. that is useful for encapsulation of dry powders in dissolvable polymeric patches for transdermal delivery and in quick dissolve tablets and films for oral delivery.
PBV is faster and less expensive than freeze-drying for producing thermostable vaccines, and PBV potentially allows for execution of barrier aseptic drying because during the PBV primary drying step the water vapor pressure above the specimen is 20 or more times higher than that during freeze-drying because PBV is performed at higher temperatures.